Lamellar bodies (LBs) are the secretory granules of the type 2 alveolar epithelial cell (AT2) through which surfactant packaging and secretion are regulated. Our previous work elucidated how lamellar bodies are formed, maintained, and utilized. An antibody identifying a unique protein of the lamellar body membrane, LBM180, was developed. Mass spectrometric sequencing showed LBM180 is identical to the ATP-Binding Cassette protein, ABCA3. This study was the first to identify ABCA3 as a lamellar body membrane, protein and to suggest its role in lamellar body biogenesis. Subsequently several genetics studies showed that mutations of the ABCA3 gene are associated with fatal surfactant deficiency and pediatric interstitial lung disease (Shulenin, 2004;Bullard, 2005) with markedly abnormal lamellar bodies in AT2 cells (Edwards, 2005). In an animal model we intend to firmly establish a causal relationship between ABCA3 mutations and lung disease and develop methods for testing therapeutics for treating these diseases. During the previous funding period we determined the location and function of ABCA3 in AT2 cells using in vitro methods. We showed: 1) ABCA3 is normally trafficked to the membranes of lamellar bodies and lysosomes and is necessary for converting lysosomes to lamellar bodies, 2) ABCA3 is necessary and sufficient for lipid accumulation, especially of choline phospholipids, in lamellar bodies and lysosomes and 3) mutations that disrupt ABCA3 expression, trafficking or function in cells could, in principal, be rescued with small molecule therapeutics. A recent development allows us to follow up these results with an additional strategy. We purchased five mice from Deltagen, a biotechnology company, and established a colony at Penn. The ABCA3-/- mice die soon after birth and their lungs fail to inflate. Aim 1: We will examine and analyze the morphology of AT2 cells from ABCA3+/+, +/- and -/- mouse lungs and measure the effect that ABCA3 expression has on LB protein and lipid content of lung cells and their secreted products to determine how differences in ABCA3 expression affect lamellar body characteristics. The LB content and PC secretion after acute changes of ABCA3 expression in cell cultures will also be measured. Aim 2: Using biochemical approaches we will determine the (lipid) substrates for ABCA3 ATPase. Aim 3: We will develop cell based screens of small molecule therapeutics for ABCA3 mutations with trafficking and folding defects. Further we will develop animal models of neonatal RDS and interstitial lung disease by generating knock-in mice with ABCA3 mutations and transgenic mice expressing conditionally controlled mutant or wild type ABCA3 in a wild type or ABCA3 KO background.